Nuclear hormone receptors are ligand-activated factors modulating the expression of target genes. How these specific regulators interact with general transcription factors is unclear. It is believed that intermediary proteins-coactivators/corepressors could promote the interaction of these receptors with the transcriptional machinery. Using the yeast-based genetic assay which analyses protein-protein interactions and thyroid hormone receptor (TR) as a bait, we screened the rat embryonic brain cDNA fusion library (GAL4-activating domain). We identified two weakly interacting proteins. One of them was an already known nuclear protein SP120 (scaffold protein)/hnRNP-U, a putative RNA/ss DNA binding protein associated with nuclear matrix and chromatin. The other cDNA (clone 6) encodes a novel protein. A. SP120/hnRNAP-U. Overexpression of hnRNP-U in transient transfection assays resulted in a repression of the reporter gene basal expression driven by a variety of polymerase (pol) II promoters. This prompted us to investigate the mechanism of hnRNP U-mediated repression. We have analyzed the effect of hnRNP U on HIV-1-LTR transcription by Rnase protection assay. Overexpression of hnRNP U in HeLa cells had no effect on initiation and elongation to position +59 (the well-characterized TAR element), but it inhibited further elongation. It has been shown that stimulation of elongation involves phosphorylation of the carboxy-terminal domain (CTD) of pol II by the TFIIH-associated kinase CAK. Thus, we examined whether hnRNp U might be associated with the pol II holoenzyme complex and, in turn, reduced processivity by affecting CTD phosphorylation. Interestingly, hnRNP U antibody coimmunoprecipitated only the nonphosphorylated form of pol II in HeLa cells extract, indicating that it associated exclusively with the nonprocessive form of pol II. In conjunction with the data that partially purified HA (hemagglutinin)-tagged hnRNP-U did not bind to the HIV promoter, these results suggest that hnRNP-U is targeted to pol II promoters via its association with pol II holoenzyme complex. In addition, preliminary results suggest that, in vitro, the immunopurified HA-hnRNP-U inhibits TFIIH-mediated phosphorylation of TBP, one of the natural substrates of TFIIH. Whether hnRNP U inhibits the TFIIH-mediated CTD phosphorylation and/or possesses intrinsic CTD phosphatase activities are under investigation. B. Clone 6. Initially, we obtained about 650 bp of this novel cDNA. This sequence was used to probe multiple tissues blot. Northern analysis revealed that this message is exclusively expressed at a high level in the rat testis and embryonic brain. Alternative splicing at the 5'end generated two mRNA forms of 2.3 and 1.5 kb. Full length cDNA clones were obtained by screening a rat testis cDNA library. DNA sequence analysis showed that this novel protein contains several repeats of a novel lecine-rich motif (LXXLL), a signature of known co-activators of a CBP family. It is likely that this protein might be a testis specific co-activator/ co-repressor of estrogen receptor. The study addressing this predication is underway.